NMDA receptor subunit NR2B plays a critical role in neurodevelopment, and has been found in animal models to influence synaptic pruning. Depletion of NR2B in radial glial neural progenitor cells (NPCs) in a rat model causes disrupted cortical lamination, though the mechanism is unknown. Patients with NR2B mutations com- monly manifest severe epilepsy and often cortical malformations suggestive of a neural migratory defect. How- ever, human models of NR2B function are entirely lacking, and treatments in vitro in in vivo in animal models have failed to translate into effective patient therapies. Two patients presenting to our Center for Neurogenetics with epileptic activity were found to carry distinct novel NR2B missense mutations, variants of uncertain signifi- cance (VUS). Fibroblasts from these patients will be used to model the effect of each VUS and create the first human model of NR2B mutation, to define the role of this protein in corticogenesis and synaptogenesis. It re- mains unknown whether and how each VUS contributes to the phenotype. Thus, in vitro analysis will be per- formed to determine the biochemical nature of each NR2B variant. Site-directed mutagenesis in NR2B cDNA plasmids and transfection into HEK-293 cells will be coupled with a cycloheximide pulse experiment to reveal each variant?s stability. Patient and wildtype (WT) fibroblasts will be reprogrammed into induced pluripotent stem cells (iPSCs), then differentiated into NPCs and glutamatergic neurons. Co-immunoprecipitation in NPC lines will determine the ability of each variant to bind NR1, requisite for the assembly of functional NMDA receptors. Mass spectroscopy will evaluate site-specific phosphorylation changes. CRISPR/Cas9 gene editing technology will be used to dissociate the mutation effects from background, by introducing each mutation into WT iPSCs, and by editing each mutation in patient lines via non-homologous end joining (NHEJ) and homology-directed repair (HDR). Neuronal cultures and cortical forebrain organoids will be derived from iPSCs to evaluate the functional effects of each mutant. Immunocytochemistry (ICC) will reveal the number of synapses per neurite, and the synaptic receptor composition. Patch-clamp recording will measure excitatory post-synaptic current (EPSC) amplitudes and action potential thresholds in each condition, and response to pharmacologic therapy, in a patient-specific model of epilepsy. NR2B mutant-derived cortical organoids will be assessed for disruption of neuronal migration in corticogenesis via immunohistochemical (IHC) staining for cortical layer markers, and cultured NPCs will be used to define the mechanism of NR2B?s role, through probing Rho GTPase activity, actin organization, and cell migration. Finally, calcium imaging will be performed on organoid slices, to evaluate net- work-level effects of NR2B mutations. These experiments will determine the impact of each novel NR2B muta- tion, to be contributed to the ClinVar database, define the mechanism of NR2B?s role in corticogenesis, establish the first human model of NR2B in synaptic development, and importantly, establish a platform for generating patient-based models of neurodevelopmental disorders for biological investigation and therapeutic assessment.